Abstract
Voltage-clamp fluorometry was performed with a cysteine-deprived mutant of rat organic cation transporter 1 (rOCT1) in which Phe483 in transmembrane α-helix (TMH) 11 close to the extracellular surface was replaced by cysteine and labeled with tetramethylrhodamine-6-maleimide. Potential-dependent fluorescence changes were observed that were sensitive to presence of substrates choline, tetraethylammonium (TEA), and 1-methyl-4-phenylpyridinium (MPP) and of the nontransported inhibitor tetrabutylammonium (TBuA). Using potential-dependent fluorescence changes as readout, one high-affinity binding site per substrate and two high-affinity binding sites for TBuA were identified in addition to the previously described single interaction sites. In a structure model of rOCT1 with an inward open cleft that was derived from a known crystal structure of lacY permease, Phe483 is close to Trp147 in TMH 2. In contrast, in a model with an outward open cleft these amino acids are far apart. After replacement of Phe483 or Trp147 by cysteine or serine, high-affinity binding of TBuA leads to inhibition of MPP or TEA uptake, whereas it has no effect on cation uptake by wild-type rOCT1. Coexisting high-affinity cation binding sites in organic cation transporters may collect low concentration xenobiotics and drugs; however, translocation including transitions between outward- and inward-oriented conformations may only be induced when a low-affinity cation binding site is loaded. We propose that cations bound to high-affinity sites may be translocated together with cations bound to low-affinity sites or that they may block the translocation mechanism.
Footnotes
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This work was supported by the Deutsche Forschungsgemeinschaft grant SFB 487/A4 (to V.G. and H.K.) and the Max-Planck-Society for the Advancement of Sciences.
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ABBREVIATIONS: OCT, organic cation transporter; OCTN, new organic cation transporter; TMH, transmembrane α-helix; r, rat; TBuA, tetrabutylammonium; TEA, tetraethylammonium; TMRM, tetramethylrhodamine-6-maleimide; MPP, 1-methyl-4-phenylpyridinium; Im, membrane current; Cm, membrane capacitance; ΔFψ, potential dependent fluorescence changes; I0.5, half-maximal current; r.m.s.d., root-mean-square deviation; MOPS, 3-(N-morpholino)propanesulfonic acid.
- Received July 19, 2007.
- Accepted October 16, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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